This invention relates to arrays, particularly polynucleotide arrays such as DNA arrays, which are useful in diagnostic, screening, gene expression analysis, and other applications.
Polynucleotide arrays (such as DNA or RNA arrays), are known and are used, for example, as diagnostic or screening tools. Such arrays include regions of usually different sequence polynucleotides arranged in a predetermined configuration on a substrate. These regions (sometimes referenced as xe2x80x9cfeaturesxe2x80x9d) are positioned at respective locations (xe2x80x9caddressesxe2x80x9d) on the substrate. The arrays, when exposed to a sample, will exhibit an observed binding pattern. This binding pattern can be detected upon interrogating the array. For example all polynucleotide targets (for example, DNA) in the sample can be labeled with a suitable label (such as a fluorescent compound), and the fluorescence pattern on the array accurately observed following exposure to the sample. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample.
Biopolymer arrays can be fabricated by depositing previously obtained biopolymers (such as from synthesis or natural sources) onto a substrate, or by in situ synthesis methods. Methods of depositing obtained biopolymers include loading then touching a pin or capillary to a surface, such as described in U.S. Pat. No. 5,807,522 or deposition by firing from a pulse jet such as an inkjet head, previously loaded with a biopolymer containing fluid, such as described in PCT publications WO 95/25116 and WO 98/41531, and elsewhere. For in situ fabrication methods, multiple different reagent droplets are deposited at a given target location in order to form the final feature (hence a probe of the feature is synthesized on the array stubstrate). The in situ fabrication methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, and described in WO 98/41531 and the references cited therein for polynucleotides. The in situ method for fabricating a polynucleotide array typically follows, at each of the multiple different addresses at which features are to be formed, the same conventional iterative sequence used in forming polynucleotides from nucleoside reagents on a support by means of known chemistry. This iterative sequence is as follows: (a) coupling a selected nucleoside through a phosphite linkage to a functionalized support in the first iteration, or a nucleoside bound to the substrate (i.e. the nucleoside-modified substrate) in subsequent iterations; (b) optionally, but preferably, blocking unreacted hydroxyl groups on the substrate bound nucleoside; (c) oxidizing the phosphite linkage of step (a) to form a phosphate linkage; and (d) removing the protecting group (xe2x80x9cdeprotectionxe2x80x9d) from the now substrate bound nucleoside coupled in step (a), to generate a reactive site for the next cycle of these steps. The functionalized support (in the first cycle) or deprotected coupled nucleoside (in subsequent cycles) provides a substrate bound moiety with a linking group for forming the phosphite linkage with a next nucleoside to be coupled in step (a). Final deprotection of nucleoside bases can be accomplished using alkaline conditions such as ammonium hydroxide, in a known manner. Reagents (nucleoside) in step (a) may be deposited as individual drops using any of the techniques previously described, while reagents for the remainder of the steps may be exposed to (flooded over) the entire substrate.
The foregoing chemistry of the synthesis of polynucleotides is described in detail, for example, in Caruthers, Science 230: 281-285, 1985; Itakura et al., Ann. Rev. Biochem. 53: 323-356; Hunkapillar et al., Nature 310: 105-110, 1984; and in xe2x80x9cSynthesis of Oligonucleotide Derivatives in Design and Targeted Reaction of Oligonucleotide Derivativesxe2x80x9d, CRC Press, Boca Raton, Fla., pages 100 et seq., U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,869,643, EP 0294196, and elsewhere The phosphoramidite and phosphite triester approaches are most broadly used, but other approaches include the phosphodiester approach, the phosphotriester approach and the H-phosphonate approach. The substrates are typically functionalized to bond to the first deposited monomer. Suitable techniques for functionalizing substrates with such linking moieties are described, for example, in Southern, E. M., Maskos, U. and Elder, J. K., Genomics, 13, 1007-1017, 1992. In the case of array fabrication, different monomers may be deposited at different addresses on the substrate during any one iteration so that the different features of the completed array will have different desired biopolymer sequences. One or more intermediate further steps may be required in each iteration, such as the conventional oxidation and washing steps in the case of in situ fabrication of polynucleotide arrays.
In array fabrication, the quantities of polynucleotide available, whether by deposition of previously obtained polynucleotides or by in situ synthesis, are usually very small and expensive. Additionally, sample quantities available for testing are usually also very small and it is therefore desirable to simultaneously test the same sample against a large number of different probes on an array. These conditions require use of arrays with large numbers of very small, closely spaced features. It is important in such arrays that features actually be present, that they are put down accurately in the desired target pattern, are of the correct size, and that the DNA is uniformly coated within the feature. If any of these conditions are not met within a reasonable tolerance, and the array user is not aware of deviations outside such tolerance, the results obtained from a given array may be unreliable and misleading. This of course can have serious consequences to diagnostic, screening, gene expression analysis or other purposes for which the array is being used. The present invention recognizes that when pulse jet or other drop deposition devices are used, one problem which can arise is the presence of gas bubbles in the fluid already loaded into the dispenser. Such bubbles can inhibit proper priming of the jet, causing it to misfire or not fire at all. Additionally, bubbles of varying size and quantity can be present in ejected drops resulting in non-uniform features.
It would be desirable then to provide a relatively simple means by which bubbles present in a drop dispenser during array fabrication, can be readily removed.
The present invention then, provides a method of fabricating an array of biopolymers on a substrate using a biopolymer or biomonomer fluid (such as a nucleic acid, for example DNA) and a drop dispenser. The drop dispenser has a chamber into which the fluid is loaded and an orifice communicating with the chamber and from which fluid is dispensed. The method includes when the chamber is loaded with a fluid, applying a prime pressure to the fluid which varies over a range sufficient to move fluid within the drop dispenser but insufficient to cause fluid to be dispensed from the orifice. Drops are dispensed from the dispenser to the substrate so as to form the array.
The varying prime pressure may be cycled at least once (and typically multiple times) between higher and lower pressures (as measured relative to each other). The duration of a cycle may vary, and may for example be between 0.01 to 5 seconds, or between 0.1 to 1 seconds. In one aspect, the varying prime pressure reaches a value during a cycle which is greater (or less) than ambient pressure outside the orifice. However, the prime pressure could be varied over a range which is both greater and less than the ambient pressure outside the orifice. The drop dispenser may be of various constructions. For example, a drop dispensing jet may include the chamber and an ejector (such as a piezoelectric or thermal ejector) which, when activated (typically electrically), causes a droplet to be ejected from the orifice. In any drop dispenser, the orifice may have an area, for example, of between 1 xcexcm2 to 3 mm2 (or between 30 xcexcm2 to 900 xcexcm2), and a capacity of the chamber in the range of between 1 pL to 10 nL.
The method may optionally additionally include loading the dispenser by positioning the orifice adjacent and facing a biomonomer or biopolymer containing fluid, and providing a load pressure to the chamber which is sufficient such that the fluid is drawn into the chamber through the orifice. The dispenser may then be positioned with the orifice facing the substrate. Multiple drops may be dispensed from the head so as to form an array of droplets on the substrate. In this case, the varying prime pressure will typically be applied following the loading and prior to the dispensing.
The present invention further provides an apparatus having a substrate station on which the substrate can be mounted. The apparatus may further include a drop dispenser having and a pressure source to apply a varying prime pressure, each as already described. The pressure source may be constructed so as to automatically apply the varying prime pressure. A load station may be present to receive at least one fluid sample for loading into the dispenser, and a transport system can selectively position the head facing any one of the stations. The pressure source may also be capable of providing the load pressure. A processor may be provided as a component of the apparatus. The processor directs the transport system to selectively position the head facing the load station or substrate station, and directs the pressure source to provide the load pressure when the head is facing the load station and to provide the varying prime pressure after the head has been loaded.
A computer program product comprising a computer readable storage medium carrying computer readable program code, for use with an apparatus of the present invention, is further provided. The program code when loaded into a computer of the apparatus causes the apparatus to carry out the steps of a method of the present invention. For example, this may include applying the varying prime pressure and dispensing drops.
The various aspects of the present invention can provide any one or more of the following and/or other useful benefits. For example, any required priming of the dispenser can be efficiently and simply accomplished, or gas bubbles in the loaded dispenser removed.